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Zebrafish biosensor for toxicant induced muscle hyperactivity.

Shahid M, Takamiya M, Stegmaier J, Middel V, Gradl M, Klüver N, Mikut R, Dickmeis T, Scholz S, Rastegar S, Yang L, Strähle U - Sci Rep (2016)

Bottom Line: Exposure to substances that interfere with motor function induced a dose-dependent increase of GFP intensity beginning at sub-micromolar concentrations, while washout of the chemicals reduced the level of hspb11 transgene expression.Simultaneously, these toxicants induced muscle hyperactivity with increased calcium spike height and frequency.TgBAC(hspb11:GFP) zebrafish embryos provide a quantitative measure of muscle hyperactivity and represent a robust whole organism system for detecting chemicals that affect motor function.

View Article: PubMed Central - PubMed

Affiliation: Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), Postfach 3640, D76021 Karlsruhe.

ABSTRACT
Robust and sensitive detection systems are a crucial asset for risk management of chemicals, which are produced in increasing number and diversity. To establish an in vivo biosensor system with quantitative readout for potential toxicant effects on motor function, we generated a transgenic zebrafish line TgBAC(hspb11:GFP) which expresses a GFP reporter under the control of regulatory elements of the small heat shock protein hspb11. Spatiotemporal hspb11 transgene expression in the musculature and the notochord matched closely that of endogenous hspb11 expression. Exposure to substances that interfere with motor function induced a dose-dependent increase of GFP intensity beginning at sub-micromolar concentrations, while washout of the chemicals reduced the level of hspb11 transgene expression. Simultaneously, these toxicants induced muscle hyperactivity with increased calcium spike height and frequency. The hspb11 transgene up-regulation induced by either chemicals or heat shock was eliminated after co-application of the anaesthetic MS-222. TgBAC(hspb11:GFP) zebrafish embryos provide a quantitative measure of muscle hyperactivity and represent a robust whole organism system for detecting chemicals that affect motor function.

No MeSH data available.


Related in: MedlinePlus

Dose-dependent and chemical-specific up-regulation of hspb11 transgene expression.Embryos from TgBAC(hspb11:GFP) were treated with the following chemicals: azinphosmethyl (A), propoxur (B), galanthamine (C), chlorpyrifos (D) and dibutylphthalate (E), veratridine (F), methylmercury (G), flucythrinate (H), esfenvalerate (I), methoxychlor (J) and chlorophenol (K), dibromoethane (L), dimethylphenol (M) and chlorothalonil (N). The hspb11 reporter levels were expressed in fold induction over control as a function of nominal chemical concentration (μM). Concentration dependent responses to the chemicals with up-regulation of the hspb11 reporter level (one-way ANOVA p < 0.001) were modelled using a logarithmic Gaussian model, except methylmercury, dibutylphthalate, methoxychlor and 4-chlorophenol for which a linear model was used. The hspb11 transgene level 1.5-fold increased over control is indicated by stippled horizontal lines in red. The EC1.5 dose (vertical stippled red lines) and modelled concentration-response curves (blue) were overlaid on the individual data points with mean (black circle) and standard deviation (vertical black bar) shown at each dose. The EC1.5 values of methoxychlor and dibutylphthalate in parenthesis are estimated values from linear modelling. Albeit their significant hspb11 transgene responses, flucythrinate and esfenvalerate were not efficiently modelled (one-way ANOVA, F(3, 116) = 6.1961, p = 6.0 × 10−4, F(3, 117) = 26.0963, p = 5.369 × 10−13, respectively). One-way ANOVA values for the rest are the following: azinphosmethyl, F(9, 310) = 44.3539, p = 1.121 × 10−50; propoxur, F(9, 278) = 51.1080, p = 6.485 × 10−54; galanthamine, F(7, 245) = 56.6725, p = 9.363 ×  × 10−48; chlorpyrifos, F(3, 121) = 68.4166, p = 6.111 × 10−26; di-n-butylphthalate, F(3, 121) = 25.4847, p = 7.555 × 10−13; veratridine, F(8, 280) = 29.0567, p = 8.135 × 10−33; methylmercury, F(3, 118) = 58.4852, p = 3.071 × 10−23; methoxychlor, F(3, 119) = 21.7458, p = 2.681 × 10−11; 4-chlorophenol, F(3, 107) = 12.2473, p = 5.969 × 10−7; 1,2-dibromoethane, F(3, 109) = 0.3508, p = 0.788; 2,4-dimethylphenol, F(3, 121) = 4.5156, p = 4.0 × 10−3; chlorothalonil, F(3, 117) = 2.0164, p = 0.115.
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f3: Dose-dependent and chemical-specific up-regulation of hspb11 transgene expression.Embryos from TgBAC(hspb11:GFP) were treated with the following chemicals: azinphosmethyl (A), propoxur (B), galanthamine (C), chlorpyrifos (D) and dibutylphthalate (E), veratridine (F), methylmercury (G), flucythrinate (H), esfenvalerate (I), methoxychlor (J) and chlorophenol (K), dibromoethane (L), dimethylphenol (M) and chlorothalonil (N). The hspb11 reporter levels were expressed in fold induction over control as a function of nominal chemical concentration (μM). Concentration dependent responses to the chemicals with up-regulation of the hspb11 reporter level (one-way ANOVA p < 0.001) were modelled using a logarithmic Gaussian model, except methylmercury, dibutylphthalate, methoxychlor and 4-chlorophenol for which a linear model was used. The hspb11 transgene level 1.5-fold increased over control is indicated by stippled horizontal lines in red. The EC1.5 dose (vertical stippled red lines) and modelled concentration-response curves (blue) were overlaid on the individual data points with mean (black circle) and standard deviation (vertical black bar) shown at each dose. The EC1.5 values of methoxychlor and dibutylphthalate in parenthesis are estimated values from linear modelling. Albeit their significant hspb11 transgene responses, flucythrinate and esfenvalerate were not efficiently modelled (one-way ANOVA, F(3, 116) = 6.1961, p = 6.0 × 10−4, F(3, 117) = 26.0963, p = 5.369 × 10−13, respectively). One-way ANOVA values for the rest are the following: azinphosmethyl, F(9, 310) = 44.3539, p = 1.121 × 10−50; propoxur, F(9, 278) = 51.1080, p = 6.485 × 10−54; galanthamine, F(7, 245) = 56.6725, p = 9.363 ×  × 10−48; chlorpyrifos, F(3, 121) = 68.4166, p = 6.111 × 10−26; di-n-butylphthalate, F(3, 121) = 25.4847, p = 7.555 × 10−13; veratridine, F(8, 280) = 29.0567, p = 8.135 × 10−33; methylmercury, F(3, 118) = 58.4852, p = 3.071 × 10−23; methoxychlor, F(3, 119) = 21.7458, p = 2.681 × 10−11; 4-chlorophenol, F(3, 107) = 12.2473, p = 5.969 × 10−7; 1,2-dibromoethane, F(3, 109) = 0.3508, p = 0.788; 2,4-dimethylphenol, F(3, 121) = 4.5156, p = 4.0 × 10−3; chlorothalonil, F(3, 117) = 2.0164, p = 0.115.

Mentions: AChE inhibitors impair muscle function by preventing the degradation of ACh at the neuromuscular junction. Genetic mutation of AChE function in the zebrafish severely disrupts muscle integrity as a result of muscle hyperactivity, and it also increases hspb11 expression2026. In line with these findings, a dose-responsive increase of hspb11 transgene expression was detected after exposure of embryos to the AChE inhibitors azinphosmethyl, propoxur27, galanthamine, and chlorpyrifos (Fig. 3A–D). Consistent with the reported weak AChE inhibiting effect of dibutylphthalate28, we observed only weak effects on hspb11 transgene expression in embryos exposed to this compound (Fig. 3E, Table S1). The effective concentrations that achieved 1.5-fold increase over control (EC1.5) varied from sub-micromolar to millimolar scale (Fig. 3A–E; Table S2), in good dosage agreement with impaired motility, particularly for the compounds which have effects on hspb11 expression already well below lethality as the apical endpoint (Fig. S1; Table S2).


Zebrafish biosensor for toxicant induced muscle hyperactivity.

Shahid M, Takamiya M, Stegmaier J, Middel V, Gradl M, Klüver N, Mikut R, Dickmeis T, Scholz S, Rastegar S, Yang L, Strähle U - Sci Rep (2016)

Dose-dependent and chemical-specific up-regulation of hspb11 transgene expression.Embryos from TgBAC(hspb11:GFP) were treated with the following chemicals: azinphosmethyl (A), propoxur (B), galanthamine (C), chlorpyrifos (D) and dibutylphthalate (E), veratridine (F), methylmercury (G), flucythrinate (H), esfenvalerate (I), methoxychlor (J) and chlorophenol (K), dibromoethane (L), dimethylphenol (M) and chlorothalonil (N). The hspb11 reporter levels were expressed in fold induction over control as a function of nominal chemical concentration (μM). Concentration dependent responses to the chemicals with up-regulation of the hspb11 reporter level (one-way ANOVA p < 0.001) were modelled using a logarithmic Gaussian model, except methylmercury, dibutylphthalate, methoxychlor and 4-chlorophenol for which a linear model was used. The hspb11 transgene level 1.5-fold increased over control is indicated by stippled horizontal lines in red. The EC1.5 dose (vertical stippled red lines) and modelled concentration-response curves (blue) were overlaid on the individual data points with mean (black circle) and standard deviation (vertical black bar) shown at each dose. The EC1.5 values of methoxychlor and dibutylphthalate in parenthesis are estimated values from linear modelling. Albeit their significant hspb11 transgene responses, flucythrinate and esfenvalerate were not efficiently modelled (one-way ANOVA, F(3, 116) = 6.1961, p = 6.0 × 10−4, F(3, 117) = 26.0963, p = 5.369 × 10−13, respectively). One-way ANOVA values for the rest are the following: azinphosmethyl, F(9, 310) = 44.3539, p = 1.121 × 10−50; propoxur, F(9, 278) = 51.1080, p = 6.485 × 10−54; galanthamine, F(7, 245) = 56.6725, p = 9.363 ×  × 10−48; chlorpyrifos, F(3, 121) = 68.4166, p = 6.111 × 10−26; di-n-butylphthalate, F(3, 121) = 25.4847, p = 7.555 × 10−13; veratridine, F(8, 280) = 29.0567, p = 8.135 × 10−33; methylmercury, F(3, 118) = 58.4852, p = 3.071 × 10−23; methoxychlor, F(3, 119) = 21.7458, p = 2.681 × 10−11; 4-chlorophenol, F(3, 107) = 12.2473, p = 5.969 × 10−7; 1,2-dibromoethane, F(3, 109) = 0.3508, p = 0.788; 2,4-dimethylphenol, F(3, 121) = 4.5156, p = 4.0 × 10−3; chlorothalonil, F(3, 117) = 2.0164, p = 0.115.
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f3: Dose-dependent and chemical-specific up-regulation of hspb11 transgene expression.Embryos from TgBAC(hspb11:GFP) were treated with the following chemicals: azinphosmethyl (A), propoxur (B), galanthamine (C), chlorpyrifos (D) and dibutylphthalate (E), veratridine (F), methylmercury (G), flucythrinate (H), esfenvalerate (I), methoxychlor (J) and chlorophenol (K), dibromoethane (L), dimethylphenol (M) and chlorothalonil (N). The hspb11 reporter levels were expressed in fold induction over control as a function of nominal chemical concentration (μM). Concentration dependent responses to the chemicals with up-regulation of the hspb11 reporter level (one-way ANOVA p < 0.001) were modelled using a logarithmic Gaussian model, except methylmercury, dibutylphthalate, methoxychlor and 4-chlorophenol for which a linear model was used. The hspb11 transgene level 1.5-fold increased over control is indicated by stippled horizontal lines in red. The EC1.5 dose (vertical stippled red lines) and modelled concentration-response curves (blue) were overlaid on the individual data points with mean (black circle) and standard deviation (vertical black bar) shown at each dose. The EC1.5 values of methoxychlor and dibutylphthalate in parenthesis are estimated values from linear modelling. Albeit their significant hspb11 transgene responses, flucythrinate and esfenvalerate were not efficiently modelled (one-way ANOVA, F(3, 116) = 6.1961, p = 6.0 × 10−4, F(3, 117) = 26.0963, p = 5.369 × 10−13, respectively). One-way ANOVA values for the rest are the following: azinphosmethyl, F(9, 310) = 44.3539, p = 1.121 × 10−50; propoxur, F(9, 278) = 51.1080, p = 6.485 × 10−54; galanthamine, F(7, 245) = 56.6725, p = 9.363 ×  × 10−48; chlorpyrifos, F(3, 121) = 68.4166, p = 6.111 × 10−26; di-n-butylphthalate, F(3, 121) = 25.4847, p = 7.555 × 10−13; veratridine, F(8, 280) = 29.0567, p = 8.135 × 10−33; methylmercury, F(3, 118) = 58.4852, p = 3.071 × 10−23; methoxychlor, F(3, 119) = 21.7458, p = 2.681 × 10−11; 4-chlorophenol, F(3, 107) = 12.2473, p = 5.969 × 10−7; 1,2-dibromoethane, F(3, 109) = 0.3508, p = 0.788; 2,4-dimethylphenol, F(3, 121) = 4.5156, p = 4.0 × 10−3; chlorothalonil, F(3, 117) = 2.0164, p = 0.115.
Mentions: AChE inhibitors impair muscle function by preventing the degradation of ACh at the neuromuscular junction. Genetic mutation of AChE function in the zebrafish severely disrupts muscle integrity as a result of muscle hyperactivity, and it also increases hspb11 expression2026. In line with these findings, a dose-responsive increase of hspb11 transgene expression was detected after exposure of embryos to the AChE inhibitors azinphosmethyl, propoxur27, galanthamine, and chlorpyrifos (Fig. 3A–D). Consistent with the reported weak AChE inhibiting effect of dibutylphthalate28, we observed only weak effects on hspb11 transgene expression in embryos exposed to this compound (Fig. 3E, Table S1). The effective concentrations that achieved 1.5-fold increase over control (EC1.5) varied from sub-micromolar to millimolar scale (Fig. 3A–E; Table S2), in good dosage agreement with impaired motility, particularly for the compounds which have effects on hspb11 expression already well below lethality as the apical endpoint (Fig. S1; Table S2).

Bottom Line: Exposure to substances that interfere with motor function induced a dose-dependent increase of GFP intensity beginning at sub-micromolar concentrations, while washout of the chemicals reduced the level of hspb11 transgene expression.Simultaneously, these toxicants induced muscle hyperactivity with increased calcium spike height and frequency.TgBAC(hspb11:GFP) zebrafish embryos provide a quantitative measure of muscle hyperactivity and represent a robust whole organism system for detecting chemicals that affect motor function.

View Article: PubMed Central - PubMed

Affiliation: Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), Postfach 3640, D76021 Karlsruhe.

ABSTRACT
Robust and sensitive detection systems are a crucial asset for risk management of chemicals, which are produced in increasing number and diversity. To establish an in vivo biosensor system with quantitative readout for potential toxicant effects on motor function, we generated a transgenic zebrafish line TgBAC(hspb11:GFP) which expresses a GFP reporter under the control of regulatory elements of the small heat shock protein hspb11. Spatiotemporal hspb11 transgene expression in the musculature and the notochord matched closely that of endogenous hspb11 expression. Exposure to substances that interfere with motor function induced a dose-dependent increase of GFP intensity beginning at sub-micromolar concentrations, while washout of the chemicals reduced the level of hspb11 transgene expression. Simultaneously, these toxicants induced muscle hyperactivity with increased calcium spike height and frequency. The hspb11 transgene up-regulation induced by either chemicals or heat shock was eliminated after co-application of the anaesthetic MS-222. TgBAC(hspb11:GFP) zebrafish embryos provide a quantitative measure of muscle hyperactivity and represent a robust whole organism system for detecting chemicals that affect motor function.

No MeSH data available.


Related in: MedlinePlus